Name: a protocol for functional assessment of whole-protein saturation mutagenesis libraries utilizing high-throughput sequencing
Uploaded: 2016-07-03T15:00:00.000+00:00
Duration: 11 min 36 s
Description: Watch this Scientific Journal Video about A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing at JoVE.com

R.R. acknowledges support from the National Institutes of Health (RO1EY018720-05), the Robert A. Welch Foundation (I-1366), and the Green Center for Systems Biology.

注:参见图1的协议纲要。在协议中几个步骤和试剂需要安全措施（与"注意"表示）。用本品前请咨询材料安全数据表。所有的协议步骤都在室温，除非其他指示进行。 1.准备文化传媒和板<ol><li>准备并通过高压灭菌1升纯化水100毫升超级最优肉汤（SOB; 表1）灭菌，1升的Luria-BERTANI肉汤（LB; 表2）和1升的LB琼脂（ 表3）。另外准备和消毒三种培养瓶中各含1升LB。 注意:在整个协议"水"是指高压釜灭菌的净化水; SOB，LB和LB琼脂指高压釜灭菌的解决方案。 </li><li>通过在10毫升70％乙醇中溶解0.12克盐酸四环素制备四环素的12毫克/毫升的库存。消毒使用0.2微米的过滤器和存储在4℃下避光保存。 </li><li>凉爽LB-琼脂至50℃，再加入1毫升四环素的库存（12微克/毫升四环素的终浓度）。倒入避光培养皿和凉爽的室温。保存在4℃下避光保存。 </li></ol> 2.全基因饱和突变库的构建注:引物;填妥的项目完成报告，限制性消化和结扎;与纯化的DNA样品可以贮存于-20℃。 <ol><li> 设计诱变引 <ol><li>诱变每个氨基酸位置的所有可能的氨基酸，设计出一对互补的诱变引物（有义/正向和反义/反），用于与以下准则的每个氨基酸位置: <ol><li>替换对应于所述氨基酸的密码子由NNS（其中N是所有四个核苷酸碱基的混合物和S是胞嘧啶（C）的混合物和鸟嘌呤（G））中的整洁和中心待诱变呃由每侧大约15个核苷酸侧翼。 </li><li>确保了5&#39;和3&#39;端终止于C或G和熔融温度（T m）为约70℃3。使用计算脚本NNS_PrimerDesign.m（见补充代码文件），根据这些原则来设计NNS诱变引。 </li></ol></li><li>从商业来源订购引物。为方便使用，让他们在96孔板格式合成和预先在水中稀释至50微米，具有一组包含有义诱变引物和另一个反义引物的板。 </li><li>装满水吸管盆，并使用多通道移液管在三个96孔平板95微升转移到263孔中。通过使用多通道移液器从含有有义诱变引物，以含有水板的同源井96孔板转移5微升稀释的引物20倍至2.5微米。 </li><li>重复协议的步骤2.1.3稀释反义诱变引。 </li></ol></li><li> 通过两步PCR的定点诱变NNS分库的每个氨基酸位置的合成 <ol><li>执行第一轮诱变的PCR。对于每一个诱变引用pBR322_AvrII质粒为模板和引物AatII_F或AvrII_R准备25微升PCR反应（与底漆AvrII_R配对感诱变引，并用引物AatII_F配对反义诱变引;共526 PCR的）。见材料表 AatII_F和AvrII_R序列。 <ol><li>通过加入从表4中的试剂到15毫升锥形管制备的PCR"主混合物"。转移到吸管盆。使用多道移液器以在三个96孔PCR板15微升转移到263孔中。使用多道移液器从包含经稀释的感诱变引物同源井96孔板转移10微升我n中的PCR板。 </li><li>覆盖一个96孔板密封每个PCR板。离心在200 xg离心2分钟。 </li><li>转移PCR板，以热循环，并运行下面的程序:98℃30秒; 20个循环:98℃10秒，55℃20秒，72℃1分钟; 72℃，2分钟;保持在4℃。 </li><li>重复协议步骤2.2.1.1 - 2.2.1.3对包含经稀释的反义诱变引物的96孔板中。 </li></ol></li><li>执行第二轮诱变的PCR。对于每个氨基酸位置，制备25μl的PCR，使用反应的引物AatII_F和AvrII_R，混合和稀释的第一轮诱变的PCR产物作为模板（总共263 PCR的）。 <ol><li>装满水吸管盆，并使用多通道移液管在三个96孔平板198微升转移到263孔中。 </li><li>通过使用多通道移液器以结合并稀释100倍的每个氨基酸位置上的突变PCR产物第一传输从含有从感测的诱变引物，以含有水板的同源孔所得的PCR产物的96孔PCR板1微升。然后重复用于从反义诱变的引物所得的PCR产物转移。 </li><li>通过添加从表5试剂到15毫升锥形管制备的PCR"主混合物"。转移到吸管盆。使用多道移液器以在三个96孔PCR板24微升转移到263孔中。使用多道移液器从含有该混合的96孔板转移1μl的稀释的第一轮诱变的PCR产物在PCR板的同源孔中。 </li><li>覆盖一个96孔板密封每个PCR板。离心机以大约200×g离心2分钟。转板，以热循环和运行同一程序作为协议的步骤2.2.1.3。 </li></ol></li><li>分析用凝胶电泳第二轮诱变PCR的结果。确保所有的产品都是正确的尺寸，并没有污染的产品。 <ol><li>加2ml 2X凝胶上样染料于一个吸管盆，然后使用多道移液器以在三个96孔平板6微升转移到263孔中。使用多道移液器从含有第二轮诱变的PCR产物，以含染料的96-孔板的同源孔96孔PCR板转移6微升。 </li><li>制备1.5％琼脂糖凝胶用0.2微克/毫升溴化乙啶（小心）。 </li><li>负载的DNA梯中的每一行的第一个和最后一个车道。然后用多道移液器从协议的步骤2.2.3.1装入10微升的样品。 </li><li>在100V用于在紫外透射40分钟和图像运行​​凝胶。 </li><li>重复步骤协议2.2.3.2 - 2.2.3.4，直到所有样本进行了分析。 </li></ol></li><li>精确测量使用dsDNA定量试剂NNS各分库PCR产物的浓度。 <ol><li>转让大约15毫升EB缓冲液以移液管盆。使用多道移液器以在三个96孔黑色壁，透明底的测定板49微升转移到263孔中。 </li><li>使用多道移液器向1μl的每个第二步骤PCR产物（协议步骤2.2.2）的转移到96孔测定板的同源孔中。 </li><li>通过在300微升EB缓冲液稀释的λ噬菌体DNA为2毫微克/微升制备DNA浓度标准曲线，然后造成十个两倍稀释（总11浓度）。转移50微升至从含有无试样的前一步骤的96孔测定板中的一个的行的第一11列;第十二列添加EB缓冲液50微升（试剂空白）。 </li><li>通过加入75微升的试剂的制备dsDNA定量试剂（参见材料的表 ），以15毫升锥形管，然后加入15mL的EB缓冲液中。通过颠倒混合管，然后转移到吸管盆地。避光剂。 </li><li>使用多道移液器至50μl制备的双链DNA定量试剂转移到测定板的各孔中。移液器向上和向下混合。在RT孵育板5分钟避光。 </li><li>测量用酶标仪和标准荧光波长（激发485纳米，发射520纳米; 0.1秒）各样品的荧光。 </li><li>从所有样品减去试剂空白的荧光值。产生从λ噬菌体样品的荧光测量值的标准曲线。计算使用他们各自的荧光测量和标准曲线各样品的浓度。 </li></ol></li></ol></li><li> NNS分库的克隆到载体的选择 <ol><li>混合100纳克NNS分库PCR产物的成五个NNS亚库组。按照厂家的说明书，清理使用DNA纯化试剂盒的样品，然后用DS浓度测量DNA定量试剂。 注意:每个组由沿TEM-1序列隔开大约53个连续的氨基酸的位置（NNS子库组1 - 5是由位置26 - 78 79 - 132 133 - 183 184 - 236，和237 - 290，分别;编号根据安布勒等[19]）。 </li><li>创建克隆载体每个NNS子库组。 <ol><li>根据表6制备个100微升的PCR，使用引物AvrII_F和AatII_OP1_R - AatII_OP5_R，和质粒pBR322_OP1-5作为模板（AatII_OP1_R与pBR322_OP1配对等）。 </li><li>转移到热循环，并运行下面的程序:98℃30秒; 25个循环:98℃10秒，55℃20秒，72℃1.5分钟; 72℃，2分钟;保持在4℃。见T 能材料的AatII_R和AvrII_F序列。 </li><li>制备的1％琼脂糖凝胶用0.2微克/毫升溴化乙啶（小心）。 </li><li>加入20μl6X凝胶样染料到每个PCR样品。负载DNA梯度凝胶的第一线;加载每个样品的总体积，跳过至少一个阱样本之间。 </li><li> 50分钟运行在100伏的凝胶。 </li><li>使用长波长紫外照明（警告）可视化凝胶。含PCR产物在约3500 bp的消费切片;转移到离心管分开。凝胶切片可以储存在-20℃。 </li><li>按照厂家的说明书，净化使用凝胶提取试剂盒和使用dsDNA定量试剂测量浓度的样品。 </li></ol></li><li>对于这两种NNS分库群（协议步2.3.1）和克隆载体（协议步2.3.2），设置限制按照T 能7用AatII和AvrⅡ消化酶消化。在37℃孵育1小时。按照厂家的说明书，清理使用DNA纯化试剂盒的样品，然后用双链DNA quantita浓度测量化试剂。 </li><li>下面的表8与关联限制性消化克隆载体（NNS分库组1 pBR322_OP1等），每个限制性消化NNS亚库组设置连接反应。孵育在RT 1小时。清理使用根据制造商的说明的DNA纯化试剂盒反应;洗脱的DNA与20微升的水。 </li><li>变换纯化连接反应的整体成库效率E.大肠杆菌细胞通过电。 <ol><li>解冻E.电感受大肠杆菌细胞，然后位置细胞，并在冰上纯化连接反应。 </li><li>将10微升解冻细胞纯化各连接反应，然后转移至电穿孔。电穿孔在1.8千伏。 </li><li>通过在1ml的SOB重悬回收细胞。在37℃下孵育1小时。 </li><li>重悬10μl，以990微升LB每个回收文化;散布在LB琼脂平板C 100微升ontaining 12微克/毫升四环素。孵育板O / N（〜16小时），在37℃。 </li><li>对于每一个恢复文化，准备用50毫升LB和50微升春节股票250毫升培养瓶中。转移到烧瓶剩下的约1毫升回收文化。在37℃下剧烈振荡（〜200rpm）下孵育O / N（〜16小时）。 </li></ol></li><li>计数每个平板上的菌落数。计算成功的转化作为数<img alt="式（1）" src="/files/ftp_upload/54119/54119eg1.jpg" /> ，其中<img alt="公式（2）" src="/files/ftp_upload/54119/54119eg2.jpg" />是菌落数， <img alt="公式3" src="/files/ftp_upload/54119/54119eg3.jpg" />是回收培养物体积（1,000微升）和<img alt="公式4" src="/files/ftp_upload/54119/54119eg4.jpg" />是恢复文化的镀量（1微升）。 注:要确保所有突变的完全覆盖，作为一个经验法则成功的转化的数量应该是≥1过00倍的预期的突变的数量。每个NNS分库有53〜职位，所以突变的预期数量为53的位置×32个密码子/位置≈1.7×10 3;给库大小≥100倍（≥1.7×10 5）应在每盘≥170菌落。 </li><li>根据制造商的说明，从使用质粒纯化试剂盒培养分离质粒DNA，然后使用dsDNA定量试剂测量的浓度。混合在一起100纳克质粒。这就造成最终的全蛋白饱和突变库。 </li></ol></li></ol> 3. TEM-1全蛋白饱和突变库抗生素抗性的选择<ol><li> 选择预培养制备。 <ol><li>从稀释步协议2.3.7质粒为0.5纳克/微升水和转移20微升至离心管。执行改造，重新covery，电镀，作为协议的步骤2.3.5 1微升的SOB恢复文化如前所述，除了转移到999微升LB中O / N增长</li><li>计数菌落数。为了确保所有的突变完全覆盖应该有≥100菌落，表明6≥10成功的转化（100×263×位置的密码子32 /位置≈10 6）。 </li><li>测量50ml的O / N培养的浓度。 <ol><li>加入1 ml LB到分光光度计小杯中准备的LB空白。测量分光光度计OD600。 </li><li>由900微升LB中重悬100微升稀释O / N培养10倍测量OD600。减去空白的OD600读数乘以10给O / N培养的OD600。 </li></ol></li><li>在37℃下预温从协议步骤1.1三种培养瓶中〜30分钟。稀释O / N文化OD600 = 0.1和1ml添加​​到一个烧瓶（最终OD600 = 0.001）。 ŧ他是"预选文化"。 </li><li>孵育"预选文化"，在37℃下剧烈振荡（200rpm）下。通过测量OD 600作为协议步骤3.1.3定期监测生长（这是没有必要稀释培养10倍），直到OD 600 = 0.1（〜2.5小时）。 </li><li>传送将100ml选择预培养到两个50毫升锥形管中。离心机在4000×g离心在4℃下6分钟。除去大部分上清液，并结合成一个单一的15锥形管中。重复离心并删除所有上清。储存于-20℃。 </li></ol></li><li> 选择氨苄青霉素抗性。 <ol><li>而选择预培养是培养，通过在10ml水中溶解0.5克钠氨苄青霉素制备安培的50毫克/毫升的库存在水中。消毒使用0.2微米的过滤器和储存在4℃。 </li><li>其它两个瓶，添加四环素至12微克/毫升的最终浓度和选择预培养这种塔的体积<html> t是最终OD600 = 0.001。一个瓶中，加入1毫升安培，为50微克/毫升的终浓度 - 这就是"选择文化"。 </li><li>孵育培养物在37℃下剧烈振荡（200rpm）下。监视这在之前步骤中加入无氨苄青霉素的培养物的生长，直至OD 600 = 0.1（〜2.5小时）。此时，也测量选择培养的OD 600。 </li><li>除以选择培养的OD600为0.1，并加入100ml繁殖。在4℃转移此体积（〜400毫升）至50ml锥形管中并离心以4,000 xg离心6分钟。除去大部分上清液，并结合成一个单一的15锥形管中。重复离心并删除所有上清。 </li><li>根据制造商的说明，从预先选择（协议步骤3.1.6）和选择（协议步骤3.2.4）培养细胞沉淀分离质粒DNA，然后使用dsDNA定量试剂测量的浓度。 </li></ol></li></ol>TLE"&gt; 4。高通量测序确定突变的健身效果<ol><li> 用于高通量测序样品的制备 <ol><li>准备25微升的PCR，以解复用正交引物NNS亚库组<ol><li>根据表9准备PCR主混合物;转移23微升到十PCR管。 </li><li>加入1微升0.5毫微克/微升纯化的质粒DNA从选择预培养到PCR管1 - 5和从选择培养至铝管6 - 10。 </li><li>混合在一起为50微米向正交引OP1_F 50微升 - OP5_F与各自的反向正交引OP1_R - OP5_R。以相同的顺序，传送1微升到PCR管1 - 5和6 - OP5_F OP1_R和- - OP5_R 材料为OP1_F序列10. 见表 。 </li><li>转移PCR管到热循环。运行下面的程序:98℃30秒; 20个循环:98℃10秒，55℃;℃20秒，72℃1.5分钟; 72℃，2分钟;保持在4℃。 </li></ol></li><li>准备25微升的PCR来隔离每个NNS子库组<ol><li>稀释100倍转增1微升每个分离PCR管中，加入99微升水从协议的步骤4.1.1.4十个项目完成报告。混合，然后吸取了99微升并丢弃。 </li><li>混合在一起的50μM正向引Group1_F 50微升 - Group5_F与相应的反向引Group1_R - Group5_R。以相同的顺序，换乘1微升至PCR管1 - 5,6 - Group5_F和Group1_R - - Group5_R 材料为Group1_F序列10.请见下表 。 </li><li>根据表9准备PCR预混液，23微升转移到各个PCR管。转移PCR管以热循环;运行同一程序作为协议的步骤2.2.1.3。 </li></ol></li><li>进行最后的25微升的PCR添加索引序列<ol><li>迪转增1微升每个分离PCR管中，加入99微升的水琵琶100倍，从协议的步骤4.1.2.3十个项目完成报告。混合，然后吸取了99微升并丢弃。 </li><li>根据表9准备PCR预混液，23微升转移到各个PCR管。 </li><li>分别505_F - 对于管与模板从NNS分库第1-5组，始发501_F转每管正向引物0.5微升。为与来自预选和选择培养得到的模板管，传送每管0.5微升分别反向引701_R和702_R。对于501_F序列见材料表 - 505_F和701_R和702_R。 </li><li>转移PCR管的热循环，并从步骤2.2.1.3运行程序。 </li></ol></li><li>混合和样品净化<ol><li>使用根据制造商的说明一dsDNA定量试剂测量的浓度。混合100纳克每PCR产物的intOA单离心管中。 </li><li>制备2％的琼脂糖凝胶，0.2微克/毫升溴化乙啶（小心）。 </li><li> 6×凝胶上样染料加入到该混合的PCR产物的样品。负载DNA梯度凝胶的第一线;装载样品的整个体积。 </li><li> 50分钟运行在100伏的凝胶。使用长波长紫外照明（警告）可视化凝胶。包含〜360 bp的PCR产物消费税切片;转移到离心管。凝胶切片可以储存在-20℃。 </li><li>按照厂家的说明书，使用凝胶提取试剂盒纯化样品和使用dsDNA定量试剂测量浓度。这是用于高通量测序的最终样品。 </li></ol></li><li>在高通量测序平台的序列（见本协议用 ​​于平台的材料表 ）。 <ol><li>计算样品浓度纳米， <img alt="公式5" src="/files/ftp_upload/54119/54119eg5.jpg"/&gt;，其中<img alt="公式6" src="/files/ftp_upload/54119/54119eg6.jpg" />是在毫微克/微升样品的浓度和<img alt="公式7" src="/files/ftp_upload/54119/54119eg7.jpg" />是样品的DNA（〜360 bp）的序列长度。稀释样品的EB缓冲液4纳米。 </li><li>按照制造商的说明变性样品稀释至晚上9时在杂交缓冲。 </li><li>负载600微升样品放入试剂盒。以下顺序制造商的说明和屏幕上的提示进行操作。 </li></ol></li></ol></li><li> 测序数据的分析 <ol><li>下载Flash（短的快速长度调整读取）20，放入文件夹与测序，获得fastq.gz文件一起。 </li><li>使用Flash加入对应的配对末端的fastq.gz文件中读取每对指数（正向和反向读取的预选和选择的文化NNS各分库组）。 <o升&gt; <li>打开命令提示符，将目录更改为在协议步4.2.1文件夹中。使用命令读取加入每对:闪&lt;mates1.fastq.gz&gt; &lt;mates2.fastq.gz&gt;，其中mates1.fastq.gz和mates2.fastq.gz是包含着文件和反转分别读取。 </li></ol></li><li>加入每对读取后，将out.extendedFrags.fastq输出文件到不同的文件夹从预选或选择文化的结果。根据NNS亚库组其所对应（ 即 ，1.fastq，2.fastq等）重命名out.extendedFrags.fastq输出文件。 </li><li>运行计算脚本NNS_DataAnalyzer.m从每个文件夹（见补充代码文件），以计算每个单氨基酸突变的计数和计数为野生型，对于每个NNS子文库基。 </li><li>计算的健身效果<img alt="式（8）" src="/files/ftp_upload/54119/54119eg8.jpg" />每个突变<img a所述="等式9"SRC ="/文件/ ftp_upload / 54119 / 54119eg9.jpg"/&gt;在每个位置<img alt="10式" src="/files/ftp_upload/54119/54119eg10.jpg" />作为碱10对数的选择获得的计数的比值（ <img alt="11式" src="/files/ftp_upload/54119/54119eg11.jpg" /> ）与预先选择（ <img alt="12式" src="/files/ftp_upload/54119/54119eg12.jpg" /> ）条件下，相对于野生型: <img alt="13式" src="/files/ftp_upload/54119/54119eg13.jpg" /> 。 </li></ol></li></ol>

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The authors declare they have no competing financial interests

这里一个协议被用于进行全蛋白饱和诱变文库的功能评估，使用高通量测序技术说明。该方法的一个重要方面是在克隆过程中使用的正交引物。简单地说，每个氨基酸位置由诱变PCR随机，并混合在一起成为其组合序列长度由高通量测序收容位置的基团。这些基团被克隆入含有成对正交引发位点，混合在一起，并进行选择的质粒载体，然后使用正交引物解复用，并随后深测序。由于突变测序中阅读局限于长度的限制，这种方式能够最大程度的有效读取数含突变的大小比基因测序读长长度。此外，该技术允许同时或"一批次"选择整个mutatio的纳尔库，减少了工作量以及该突变经历不同水平的选择的可能性。实际上，在该协议在很大程度上关注组织中的关键步骤:在克隆过程（协议步骤2.3）必须确保的诱变PCR产物的正确混合成团并随后将其克隆到正确正交引发位向量;制备样品的测序（协议步骤4.1）中，正确的正交的引物，以及作为引物，以每个NNS子文库组的隔离，并添加索引序列，必须使用。 该协议的三个主要步骤: - 文库构建，选择和测序 - 可以在几个方面进行修改。在文库构建人们可以引入使用多种技术的突变，例如，通过易错PCR，或通过构建使用由在替代核苷酸的一小部分掺杂合成的寡核苷酸的基因第21人们可以构建文库以包括该蛋白质的片段（ 即，NNS亚文库基），或在替代的基因型背景22内双键或高阶突变。重要的是，所有修改的文库构建步骤但是应该满足的顺序和测序中的突变的位置之间的对应关系读长度被维持的标准。因此，这个标准排除了对跨多个蛋白质的突变综合研究协议的应用。修改协议的第二部分包括交替选择条件:（ 如 ，温度，营养物水平）不同β内酰胺类型（或组合）和浓度，外部应力的条件下，主机类型（ 例如，不同类型的细菌），或不同的采样时间（几小时到几天）。例如，在以前的工作中，我们检查了所有单个氨基酸突变的健身效果在TEM-1不同浓度的氨苄青霉素的下，第三代头孢菌素噻肟13下。至于该协议的第三步骤中，我们目前不推荐从这里使用的测序平台的选择偏离（参见材料的数据表）。尽管测序读长的确是目前不再在其他平台上，读取数获得目前低得多;一般地，可确定一个突变的效果的准确度成正比读取得到的数量（见协议步骤4.2.5方程）。 主要是为了简单起见，该协议使用TEM-1β内酰胺酶作为模型系统，但是这里描述的方法可以扩展到其他系统为其中高通量选择或筛选测定是在适当位置。构建这样的测定然而往往不是那么简单:首先，对于基因（突变）和蛋白质的条块战略一起，必须建立例如在小区内，液滴（如在微流体平台），或通过噬菌体展示。第二，也是最重要的是，蛋白质功能和可选择的表型，或健身之间的量化的连接，必须建立。为参与代谢或抗生素抗性的酶，细胞的养分辍学或抗生素的培养基中生长的能力通常是酶活性的直接函数。更合成方法可以在其他系统通过连接蛋白-蛋白结合亲和性的报道基因可用于，例如（ 例如 ，荧光蛋白）在细菌中表达或酵母11,23，或使用在一个微流体系统24一个荧光酶底物。最后，这样的测定必须是可伸缩的，以解决全蛋白诱变文库的大小。 总之，对于全蛋白饱和诱变文库的功能评估高通量基于测序的方法是DESCRIBED在这里。中央的方式是诱变文库的沿基因区段内的结构，和正交底漆条形码的利用率来标记，用于多路复用和去多路复用的库中的每个段。我们预计，该协议可以很容易地应用到其中一个适当的高通量选择或屏幕已开发的其他蛋白质。

诱变早已在实验室用于研究生物系统及其演进的属性，并产生突变蛋白质或微生物与增强的或新的功能。虽然早期的方法在其上产生生物随机突变的方法依赖，重组DNA技术的出现使研究人员能够引入位点特异性方式与DNA选择的变化， 即 ，位点定向诱变1,2。与目前的技 ​​术，一般是在聚合酶链反应（PCR），使用诱变寡核苷酸，它是相对容易的在给定基因3,4-创建并评估突变的小的数字（ 例如 ，点突变）。这是更为困难然而，当目标的办法，例如，所有可能的单站点的创建和评估（或更高阶）的突变。 虽然很多已经从早期的研究试图评估大量m的教训在基因utations，所采用的技术往往是费力的，例如要求每个突变评估独立使用的废话抑制5-7株，或在自己的能力量化，由于Sanger测序8的低测序深度有限。在这些研究中所使用的技术已在很大程度上被利用高通量测序技术方法9-12取代。这些概念简单的方法意味着创建包括了大量的突变库，库中经受了功能的屏幕或选择，然后深测序（ 即 ，&gt; 10 6排序的顺序读取时）之前获得图书馆，选择后。以这种方式，大量的突变的表型或健身效果，表示为在每个突变体的群体频率的变化，可以同时和更定量评估。 我们之前推出了SIMP（ 即 ，全蛋白饱和诱变文库）用于评估蛋白质的所有可能的单氨基酸突变的文库，适用于基因的一个长度比测序长文件的方法读取长度11,13:首先，每个氨基酸位置是随机通过位点定向诱变的PCR。在此过程中，该基因被分成与由测序平台收纳总长度连续位置组成的组。诱变的PCR产物为每个组，然后组合，并且每个基团独立地进行选择和高通量测序。通过保持序列和测序读长在基因突变的位置之间的对应关系，这种方法也有最大化的测序深度的优点:当一个可以简单地序列中短窗这样的库，不要拆开成团（ 例如 ，通过一个标准的猎枪。测序方法），最读取得到的将是野生型和由此m个浪费测序吞吐量ajority（ 例如，在100个氨基酸（300 bp）的窗户测序一个500个氨基酸的蛋白的一个全蛋白饱和诱变文库，以最低80％的读出将是野生型序列）。 这里，提出了一种协议，它利用高通量测序为全蛋白饱和诱变文库的功能评估，使用上述方法（ 图1中所述）。重要的是，我们引入的正交的引物的使用量在库中克隆过程条形码每个序列组，这允许它们被复用到一个库，同时进行筛选或选择，然后解复用为深度测序。由于序列组不进行选择独立，这减少了工作量，并确保各突变经历选择的相同水平。 TEM-1β内酰胺酶，其赋予对高层次性的酶β内酰胺类抗生素（ 如氨苄青霉素）的细菌被用作模型系统14-16。的协议是用于TEM-1在大肠杆菌中一个全蛋白饱和诱变文库的评估中描述大肠杆菌下，在对一个临床剂量氨苄青霉素（50微克/毫升）17,18的近似的血清水平的选择。

<table><tbody><tr><td>Typtone</td><td>Research Products Intl. Corp.</td><td>T60060-1000.0</td><td></td></tr><tr><td>Yeast extract</td><td>Research Products Intl. Corp.</td><td>Y20020-500.0</td><td></td></tr><tr><td>Sodium chloride</td><td>Fisher Scientific</td><td>BP358-212</td><td></td></tr><tr><td>Potassium chloride</td><td>Sigma-Aldrich</td><td>P9333-500G</td><td></td></tr><tr><td>Magnesium sulfate</td><td>Sigma-Aldrich</td><td>M7506-500G</td><td></td></tr><tr><td>Agar</td><td>Fisher Scientific</td><td>BP1423-500</td><td></td></tr><tr><td>Tetracycline hydrochloride</td><td>Sigma-Aldrich</td><td>T7660-5G</td><td></td></tr><tr><td>Petri plates</td><td>Corning</td><td>351029</td><td></td></tr><tr><td>MATLAB&amp;nbsp;</td><td>Mathworks</td><td>http://www.mathworks.com/products/matlab/</td><td></td></tr><tr><td>Oligonucleotide primers</td><td>Integrated DNA Technologies</td><td>https://www.idtdna.com/pages/products/dna-rna/custom-dna-oligos</td><td>25 nmol scale, standard desalting</td></tr><tr><td>pBR322_AvrII</td><td>available upon request</td><td></td><td>pBR322 plasmid modified to contain AvrII restriction site downstream of the TEM-1 gene</td></tr><tr><td>pBR322_OP1 &amp;ndash; pBR322_OP5</td><td>available upon request</td><td></td><td>five modified pBR322 plasmids each containing a pair of orthogonal priming sites</td></tr><tr><td>Q5 high-fidelity DNA polymerase</td><td>New England Biolabs</td><td>M0491L</td><td>includes 5x PCR buffer and PCR additive (GC enhancer)</td></tr><tr><td>15 ml conical tube</td><td>Corning</td><td>430025</td><td></td></tr><tr><td>Multichannel pipettes (Eppendorf ResearchPlus)</td><td>Eppendorf</td><td></td><td></td></tr><tr><td>PCR plate, 96 well</td><td>Fisher Scientific</td><td>14230232</td><td></td></tr><tr><td>96 well plate seal</td><td>Excel Scientific</td><td>F-96-100</td><td></td></tr><tr><td>Veriti 96-well thermal cycler</td><td>Applied Biosystems</td><td>4375786</td><td></td></tr><tr><td>6x gel loading dye</td><td>New England Biolabs</td><td>B7024S</td><td></td></tr><tr><td>Agarose</td><td>Research Products Intl. Corp.</td><td>20090-500.0</td><td></td></tr><tr><td>Ethidium bromide</td><td>Bio-Rad</td><td>161-0433</td><td></td></tr><tr><td>UV transilluminator (FOTO/Analyst ImageTech)</td><td>Fotodyne Inc.</td><td>http://www.fotodyne.com/content/ImageTech_gel_documentation</td><td></td></tr><tr><td>EB buffer</td><td>Qiagen</td><td>19086</td><td></td></tr><tr><td>96-well black-walled, clear bottom assay plates</td><td>Corning</td><td>3651</td><td></td></tr><tr><td>Lambda phage DNA</td><td>New England Biolabs</td><td>N3011S</td><td></td></tr><tr><td>PicoGreen dsDNA reagent</td><td>Invitrogen</td><td>P7581</td><td>dsDNA quantitation reagent, used in protocol step 2.2.4</td></tr><tr><td>Victor 3 V microplate reader</td><td>PerkinElmer</td><td></td><td></td></tr><tr><td>DNA purification kit</td><td>Zymo Research</td><td>D4003</td><td></td></tr><tr><td>Microcentrifuge tubes</td><td>Corning</td><td>3621</td><td></td></tr><tr><td>Long-wavelength UV illuminator</td><td>Fisher Scientific</td><td>FBUVLS-80</td><td></td></tr><tr><td>Agarose gel DNA extraction buffer</td><td>Zymo Research</td><td>D4001-1-100</td><td></td></tr><tr><td>AatII</td><td>New England Biolabs</td><td>R0117S</td><td></td></tr><tr><td>AvrII</td><td>New England Biolabs</td><td>R0174L</td><td></td></tr><tr><td>T4 DNA ligase</td><td>New England Biolabs</td><td>M0202S</td><td></td></tr><tr><td>EVB100 electrocompetent E. coli</td><td>Avidity</td><td>EVB100</td><td></td></tr><tr><td>Electroporator (E. coli Pulser)</td><td>Bio-Rad</td><td>1652102</td><td></td></tr><tr><td>Electroporation cuvettes</td><td>Bio-Rad</td><td>165-2089</td><td></td></tr><tr><td>Spectrophotometer (Ultrospec 3100 pro)</td><td>Amersham Biosciences</td><td>80211237</td><td></td></tr><tr><td>50 ml conical tubes</td><td>Corning</td><td>430828</td><td></td></tr><tr><td>Plasmid purification kit</td><td>Macherey-Nagel</td><td>740588.25</td><td></td></tr><tr><td>8 well PCR strip tubes</td><td>Axygen</td><td>321-10-551</td><td></td></tr><tr><td>Qubit dsDNA HS assay kit</td><td>Invitrogen</td><td>Q32854</td><td>dsDNA quantitation reagent</td></tr><tr><td>Qubit assay tubes</td><td>Invitrogen</td><td>Q32856</td><td></td></tr><tr><td>Qubit fluorometer</td><td>Invitrogen</td><td>Q32866</td><td></td></tr><tr><td>Ampicillin sodium salt</td><td>Akron Biotechnology</td><td>50824296</td><td></td></tr><tr><td>MiSeq reagent kit v2 (500 cycles)</td><td>Illumina</td><td>MS-102-2003</td><td></td></tr><tr><td>MiSeq desktop sequencer</td><td>Illumina</td><td>http://www.illumina.com/systems/miseq.html</td><td>alternatively, one could sequence on Illumina HiSeq platform</td></tr><tr><td>FLASh software</td><td>John Hopkins University - open source</td><td>http://ccb.jhu.edu/software/FLASH/</td><td>software to merge paired-end reads from next-generation sequencing data</td></tr><tr><td>AatII_F</td><td></td><td></td><td>GATAATAATGGTTTCTTAGACG&lt;br/&gt;TCAGGTGGC</td></tr><tr><td>AvrII_R</td><td></td><td></td><td>CTTCACCTAGGTCCTTTTAAAT&lt;br/&gt;TAAAAATGAAG</td></tr><tr><td>AvrII_F</td><td></td><td></td><td>CTTCATTTTTAATTTAAAAGGA&lt;br/&gt;CCTAGGTGAAG</td></tr><tr><td>AatII_OP1_R</td><td></td><td></td><td>ACCTGACGTCCGTATTTCAAC&lt;br/&gt;
TGTCCGGTCTAAGAAACCATT&lt;br/&gt;
ATTATCATGACATTAAC</td></tr><tr><td>AatII_OP2_R</td><td></td><td></td><td>ACCTGACGTCCGCTCACGGA&lt;br/&gt;
GTGTACTAATTAAGAAACCATT&lt;br/&gt;
ATTATCATGACATTAAC</td></tr><tr><td>AatII_OP3_R</td><td></td><td></td><td>ACCTGACGTCGTACGTCTGA&lt;br/&gt;
ACTTGGGACTTAAGAAACCA&lt;br/&gt;
TTATTATCATGACATTAAC</td></tr><tr><td>AatII_OP4_R</td><td></td><td></td><td>ACCTGACGTCCCGTTCTCGAT&lt;br/&gt;
ACCAAGTGATAAGAAACCATT&lt;br/&gt;
ATTATCATGACATTAAC</td></tr><tr><td>AatII_OP5_R</td><td></td><td></td><td>ACCTGACGTCGTCCGTCGGA&lt;br/&gt;
GTAACAATCTTAAGAAACCAT&lt;br/&gt;
TATTATCATGACATTAAC</td></tr><tr><td>OP1_F</td><td></td><td></td><td>GACCGGACAGTTGAAATACG</td></tr><tr><td>OP1_R</td><td></td><td></td><td>CGACGTACAGGACAATTTCC</td></tr><tr><td>OP2_F</td><td></td><td></td><td>ATTAGTACACTCCGTGAGCG</td></tr><tr><td>OP2_R</td><td></td><td></td><td>AGTATTAGGCGTCAAGGTCC</td></tr><tr><td>OP3_F</td><td></td><td></td><td>AGTCCCAAGTTCAGACGTAC</td></tr><tr><td>OP3_R</td><td></td><td></td><td>GAAAAGTCCCAATGAGTGCC</td></tr><tr><td>OP4_F</td><td></td><td></td><td>TCACTTGGTATCGAGAACGG</td></tr><tr><td>OP4_R</td><td></td><td></td><td>TATCACGGAAGGACTCAACG</td></tr><tr><td>OP5_F</td><td></td><td></td><td>AGATTGTTACTCCGACGGAC</td></tr><tr><td>OP5_R</td><td></td><td></td><td>TATAACAGGCTGCTGAGACC</td></tr><tr><td>Group1_F</td><td></td><td></td><td>ACACTCTTTCCCTACACGAC&lt;br/&gt;
GCTCTTCCGATCTNNNNNGC&lt;br/&gt;
ATTTTGCCTACCGGTTTTTGC</td></tr><tr><td>Group1_R</td><td></td><td></td><td>GTGACTGGAGTTCAGACGTG&lt;br/&gt;
TGCTCTTCCGATCTNNNNNTC&lt;br/&gt;
TTGCCCGGCGTCAAC</td></tr><tr><td>Group2_F</td><td></td><td></td><td>ACACTCTTTCCCTACACGAC&lt;br/&gt;
GCTCTTCCGATCTNNNNNGA&lt;br/&gt;
ACGTTTTCCAATGATGAGCAC</td></tr><tr><td>Group2_R</td><td></td><td></td><td>GTGACTGGAGTTCAGACGTG&lt;br/&gt;
TGCTCTTCCGATCTNNNNNGT&lt;br/&gt;
CCTCCGATCGTTGTCAGAAG</td></tr><tr><td>Group3_F</td><td></td><td></td><td>ACACTCTTTCCCTACACGAC&lt;br/&gt;
GCTCTTCCGATCTNNNNNAG&lt;br/&gt;
TAAGAGAATTATGCAGTGCTGCC</td></tr><tr><td>Group3_R</td><td></td><td></td><td>GTGACTGGAGTTCAGACGTG&lt;br/&gt;
TGCTCTTCCGATCTNNNNNTC&lt;br/&gt;
GCCAGTTAATAGTTTGCGC</td></tr><tr><td>Group4_F</td><td></td><td></td><td>ACACTCTTTCCCTACACGAC&lt;br/&gt;
GCTCTTCCGATCTNNNNNCC&lt;br/&gt;
AAACGACGAGCGTGACAC</td></tr><tr><td>Group4_R</td><td></td><td></td><td>GTGACTGGAGTTCAGACGTG&lt;br/&gt;
TGCTCTTCCGATCTNNNNNGC&lt;br/&gt;
AATGATACCGCGAGACCC</td></tr><tr><td>Group5_F</td><td></td><td></td><td>ACACTCTTTCCCTACACGAC&lt;br/&gt;
GCTCTTCCGATCTNNNNNCG&lt;br/&gt;
GCTGGCTGGTTTATTGC</td></tr><tr><td>Group5_R</td><td></td><td></td><td>GTGACTGGAGTTCAGACGTG&lt;br/&gt;
TGCTCTTCCGATCTNNNNNTAT&lt;br/&gt;
ATGAGTAAACTTGGTCTGACAG</td></tr><tr><td>501_F</td><td></td><td></td><td>AATGATACGGCGACCACCGA&lt;br/&gt;
GATCTACACTATAGCCTACAC&lt;br/&gt;
TCTTTCCCTACACGAC</td></tr><tr><td>502_F</td><td></td><td></td><td>AATGATACGGCGACCACCGA&lt;br/&gt;
GATCTACACATAGAGGCACA&lt;br/&gt;
CTCTTTCCCTACACGAC</td></tr><tr><td>503_F</td><td></td><td></td><td>AATGATACGGCGACCACCGA&lt;br/&gt;
GATCTACACCCTATCCTACAC&lt;br/&gt;
TCTTTCCCTACACGAC</td></tr><tr><td>504_F</td><td></td><td></td><td>AATGATACGGCGACCACCGA&lt;br/&gt;
GATCTACACGGCTCTGAACA&lt;br/&gt;
CTCTTTCCCTACACGAC</td></tr><tr><td>505_F</td><td></td><td></td><td>AATGATACGGCGACCACCGA&lt;br/&gt;
GATCTACACAGGCGAAGACA&lt;br/&gt;
CTCTTTCCCTACACGAC</td></tr><tr><td>701_R</td><td></td><td></td><td>CAAGCAGAAGACGGCATAC&lt;br/&gt;
GAGATCGAGTAATGTGACTG&lt;br/&gt;
GAGTTCAGACGTG</td></tr><tr><td>702_R</td><td></td><td></td><td>CAAGCAGAAGACGGCATAC&lt;br/&gt;
GAGATTCTCCGGAGTGACTG&lt;br/&gt;
GAGTTCAGACGTG</td></tr></tbody></table>

a protocol for functional assessment of whole-protein saturation mutagenesis libraries utilizing high-throughput sequencing

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel sequencing technology have opened up the possibility of assessing the functional or fitness effects of large numbers of mutations simultaneously. Here, we present a protocol for experimentally determining the effects of all possible single amino acid mutations in a protein of interest utilizing high-throughput sequencing technology, using the 263 amino acid antibiotic resistance enzyme TEM-1 &#946;-lactamase as an example. In this approach, a whole-protein saturation mutagenesis library is constructed by site-directed mutagenic PCR, randomizing each position individually to all possible amino acids. The library is then transformed into bacteria, and selected for the ability to confer resistance to &#946;-lactam antibiotics. The fitness effect of each mutation is then determined by deep sequencing of the library before and after selection. Importantly, this protocol introduces methods which maximize sequencing read depth and permit the simultaneous selection of the entire mutation library, by mixing adjacent positions into groups of length accommodated by high-throughput sequencing read length and utilizing orthogonal primers to barcode each group. Representative results using this protocol are provided by assessing the fitness effects of all single amino acid mutations in TEM-1 at a clinically relevant dosage of ampicillin. The method should be easily extendable to other proteins for which a high-throughput selection assay is in place.

对于含有正交引发位点的五个修饰的pBR322质粒的质粒图谱（pBR322_OP1 - pBR322_OP5）示于图2A。为了测试正交的引物是否是特定使用每对正交引物单独进行的PCR，与所有五个pBR322_OP1-5质粒一起，或与所有质粒减去匹配正交引物对中的质粒。当被包括在匹配的质粒仅获得正确的产物，并在它的缺失（ 图2B），没有得到任何大小的产物。 以下在本文中（ 图1）中描述的方案进行的代表性实验。以下处理（协议第4.2节），6.2×10 6从预选条件的读取和6.3×10 6从50微克/毫升氨苄青霉素选择conditi读上获得。对于每个所获得的计数氨基酸从预选条件酸突变显示一个特征数正态分布（ 图3A）13。至少一个从选择预培养测序计数突变98.9％（58没有计数），和大于100的计数为91.2％（有465小于100个计数）获得。 图3B示出了相对的健身效果（），用于在TEM-1的各位置的每个突变;在图3C中示出的分布。下，在50μg/ ml的氨苄青霉素选择，大多数突变具有中性或接近中性的健身效果（≈0，对应于图3B的白色像素和图3C中的大峰）。在该浓度下突变的一小部分对健身实质性影响（&lt;&lt; 0，对应于图3B和图3C中左侧尾蓝色像素）; EXP<html> ectedly，这其中就包括高度保守的活性位点残基（S70，K73，S130，D131，N132，K234和G236）13,14内突变。与此相反，一些突变显着地增加健身超过该TEM-1（&gt;&gt; 0，对应于图3B红色像素），可以预料，因为TEM-1是在氨苄青霉素水解高效（ <img alt="14式" src="/files/ftp_upload/54119/54119eg14.jpg" /> ≈10 7 M -1 -1）14。 <img alt="图1" src="/files/ftp_upload/54119/54119fig1.jpg" /> 图1.根据氨苄青霉素选择TEM-1β内酰胺酶全蛋白饱和诱变协议的概要。如在协议中所述操作以粗体显示。编号协议的步骤在左边显示，以供参考的主要内容。K"&gt;点击此处查看该图的放大版本。 <img alt="图2" src="/files/ftp_upload/54119/54119fig2.jpg" /> 图2.正交引发位质粒载体验证。 （A）质粒图包含正交引发位点（pBR322_OP1 - pBR322_OP5）五修饰的pBR322质粒。正交引发位点的位置和方向指示。几个酶切位点位置标记;在TEM-1全蛋白饱和诱变文库（其包括整个TEM-1基因和启动子）克隆在中间的的AatII和AvrII位限制位点。缩写:TET四环素抗性基因，ORI复制起点（B）的每对正交引物（OP1-OP5）在含有所有五个pBR322_OP1-5质粒（+），或与所有质粒减去各质粒的PCR进行了测试（。 ˗）。示出的是溴化乙锭染色的琼脂糖凝胶（1％重量/体积）装入每个PCR反应，大小通过电泳分离。预期尺寸产品是1628基点;第一道是DNA梯，相关标准尺寸显示。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54119/54119fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54119/54119fig3.jpg" /> 图3的TEM-1β内酰胺酶全蛋白饱和度实验的结果。 （A）的直方图表示计数用于从预选条件库的高通量测序获得的各氨基酸突变的分布。为了简单起见，用零计数（53突变）的突变是在50μg/ ml的氨苄青霉素的示出为具有一个的计数。所有单个氨基酸突变的（B）的健身效果在TEM-1下的选择。示出的是数据含有相对健身效果（），蓝色代表有害影响比色描绘矩阵，红与野生型有积极的影响，相对白色无健身效果。为这是从预选培养没有得到计数突变黑色。行描绘沿主序列位置和列表示突变的二十种氨基酸的一种或终止密码子（由单字母码表示，*为终止密码子）; TEM-1的二级结构在活性部位内的左侧和几个高度保守的基序在右边指示被指示（C）直方图示出的相对健身效果的分布。显示是从测序预选文化得到&gt; 100计数这些基因突变的结果。 <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/54119/54119fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <table border="1" fo:keep-together.within-page="1" fo:kEEP-与-next.within页="总是"&gt; <tbody><tr><td> 试剂 </td><td> 质量或体积 </td><td> 评论 </td></tr><tr><td> Typtone </td><td> 2克</td><td></td></tr><tr><td>酵母抽提物</td><td> 0.5克</td><td></td></tr><tr><td>氯化钠</td><td> 0.06克</td><td></td></tr><tr><td>氯化钾</td><td> 0.02克</td><td></td></tr><tr><td>硫酸镁</td><td> 0.24克</td><td></td></tr><tr><td>净化水</td><td> 100毫升</td><td></td></tr></tbody></table> 表1.超级最佳肉汤（SOB）。在准备百毫升SOB（协议步骤1.1）所使用试剂的名称和数量。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 试剂 </td><td> 大众或卷UME </td><td> 评论 </td></tr><tr><td> Typtone </td><td> 10克</td><td></td></tr><tr><td>酵母抽提物</td><td> 5克</td><td></td></tr><tr><td>氯化钠</td><td> 10克</td><td></td></tr><tr><td>净化水</td><td> 1升</td><td></td></tr></tbody></table> 表2.卢里亚BERTANI肉汤（LB），试剂名称和数量准备1升LB（协议步骤1.1）中使用。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 试剂 </td><td> 质量或体积 </td><td> 评论 </td></tr><tr><td> Typtone </td><td> 10克</td><td></td></tr><tr><td>酵母抽提物</td><td> 5克</td><td></td></tr><tr><td>氯化钠</td><td> 10克</td><td></td></tr><tr><td>洋菜</td><td> 15克</td><td></td></tr><tr><td>净化水</td><td> 1升</td><td></td></tr></tbody></table>在制备LB-琼脂（协议步骤1.1）中使用表3的LB琼脂上。试剂名称和数量。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 试剂 </td><td> 卷 </td><td> 评论 </td></tr><tr><td> 5X PCR缓冲液</td><td> 1450微升</td><td></td></tr><tr><td> PCR添加剂</td><td> 1450微升</td><td></td></tr><tr><td> 2毫米的dNTP </td><td> 725微升</td><td> 2毫米每个核苷酸</td></tr><tr><td> 50μMAatII_F或AvrII_R底漆</td><td> 145微升</td><td></td></tr><tr><td> 1纳克/微升pBR322_AvrII质粒</td><td> 145微升</td><td></td></tr><tr><td> 2单位/μLDNA聚合酶</td><td> 72.5微升</td><td></td></tr><tr><td>水</td><td> 363微升</td><td></td></tr></tbody></table> 表4.第一轮诱变PCR预混，准备主混音的第一轮诱变PCR（协议步2.2.1）试剂的名称和数量。总量足以290 25μL反应。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 试剂 </td><td> 卷 </td><td> 评论 </td></tr><tr><td> 5X PCR缓冲液</td><td> 1450微升</td><td></td></tr><tr><td> PCR添加剂</td><td> 1450微升</td><td></td></tr><tr><td> 2毫米的dNTP </td><td> 725微升</td><td> 2毫米每个核苷酸</td></tr><tr><td> 50μMAatII_F底漆</td><td> 145微升</td><td></td></tr><tr><td> 50μMAvrII_R底漆</td><td> 145微升</td><td></td></tr><tr><td> 2单位/μLDNA聚合酶</td><td> 72.5微升</td><td></td></tr><tr><td>水</td><td> 2973微升</td><td></td></tr></tbody></table> 表5.第二轮诱变PCR预混，准备主混音的第二轮诱变PCR（协议步2.2.2）试剂的名称和数量。总量足以290 25微升反应。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 试剂 </td><td> 卷 </td><td> 评论 </td></tr><tr><td> 5X PCR缓冲液</td><td> 20微升</td><td></td></tr><tr><td> PCR添加剂</td><td> 20微升</td><td></td></tr><tr><td> 2毫米的dNTP </td><td> 10微升</td><td> 2毫米每个核苷酸</td></tr><tr><td> 50μMAvrII_F底漆</td><td> 2微升</td><td></td></tr><tr><td> 50μMAatII_OP1_R - AatII_OP5_R底漆</td><td> 2微升</td><td>每个反应一个引物，用各自的质粒配对</td></tr><tr><td> 1纳克/微升pBR322_OP1-5质粒</td><td> 2微升</td><td>每个反应一个质粒</td></tr><tr><td> 2单位/μLDNA聚合酶</td><td> 1微升</td><td></td></tr><tr><td>水</td><td> 43微升</td><td></td></tr></tbody></table> 表6.克隆载体PCR检测试剂的名称和数量准备PCR使克隆载体（协议步2.3.2）。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 试剂 </td><td> 卷 </td><td> 评论 </td></tr><tr><td> 10×限制性酶缓冲液</td><td> 5微升</td><td></td></tr><tr><td> 4单位/微升AvrⅡ消化</td><td> 2.5微升</td><td></td></tr><tr><td> 20单位/微升的AatII </td><td> 0.5微升</td><td></td></tr><tr><td> DNA以限制性消化</td><td>体积500纳克</td><td></td></tr><tr><td>水</td><td>至50μl总体积</td><td></td></tr></tbody></table> 表7.限制性消化，试剂的名称和数量克隆载体和NNS分库群（协议步2.3.3）的限制性消化。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 试剂 </td><td> 卷 </td><td> 评论 </td></tr><tr><td> 10×T4 DNA连接酶缓冲</td><td> 5微升</td><td></td></tr><tr><td>纯化的限制性消化NNS亚库组DNA </td><td>体积为48纳克</td><td></td></tr><tr><td>纯化的限制性消化的克隆载体DNA </td><td>体积为52纳克</td><td></td></tr><tr><td> 400单位/微升T4 DNA连接酶</td><td> 1微升</td><td></td></tr><tr><td>水</td><td> 20微升总量</td><td></td></tr></tbody></table> 表8.连接反应试剂的名称和数量与在1限制性消化NNS子库组克隆载体的连接反应:3载体:插入片段的摩尔比（协议步骤2.3.4）。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 试剂 </td><td> 卷 </td><td> 评论 </st荣&gt; </td></tr><tr><td> 5X PCR缓冲液</td><td> 55微升</td><td></td></tr><tr><td> PCR添加剂</td><td> 55微升</td><td></td></tr><tr><td> 2毫米的dNTP </td><td> 27.5微升</td><td> 2毫米每个核苷酸</td></tr><tr><td> 2单位/μLDNA聚合酶</td><td> 2.75微升</td><td></td></tr><tr><td>水</td><td> 113微升</td><td></td></tr></tbody></table> 表9. PCR试 ​​剂样品制备用于高通量测序。试剂名称和数量准备PCR预混液用于解复用正交引物（4.1.1），隔离NNS分库群（协议步骤4.1.2）并加入索引序列（协议步4.1.3）。总量足以11 25微升反应。

Watch this Scientific Journal Video about A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing at JoVE.com

A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

我们提出了一个协议为利用高通量测序蛋白的综合单站点饱和突变库功能评估。重要的是，这种方法采用正交引物对复图书馆建设和测序。提供了使用在氨苄青霉素的临床相关剂量选择的TE​​M-1β内酰胺酶代表性结果。

经过一蛋白饱和诱变图书馆的功能评价规范利用高通量测序

Site-directed mutagenesis has long been used as a method to interrogate protein structure, function and evolution. Recent advances in massively-parallel ...

a-protocol-for-functional-assessment-whole-protein-saturation

Universidad San Sebastian

Research

JoVE Journal

Biology

10.8K 次观看.  University of Texas Southwestern Medical Center. 我们提出了一个协议为利用高通量测序蛋白的综合单站点饱和突变库功能评估。重要的是，这种方法采用正交引物对复图书馆建设和测序。提供了使用在氨苄青霉素的临床相关剂量选择的TE​​M-1β内酰胺酶代表性结果。 

视频: A Protocol for Functional Assessment of Whole-Protein Saturation Mutagenesis Libraries Utilizing High-Throughput Sequencing

Next Generation Sequencing for the Detection of Actionable Mutations in Solid and Liquid Tumors

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific molecular profiles of a tumor may predict responsiveness to therapeutic agents or provide knowledge about prognosis. At our institution a tumor genotyping program was established as part of routine clinical care, screening both hematologic and solid tumors for a wide spectrum of mutations using two next-generation sequencing (NGS) panels: a custom, 33 gene hematological malignancies panel for use with peripheral blood and bone marrow, and a commercially produced solid tumor panel for use with formalin-fixed paraffin-embedded tissue that targets 47 genes commonly mutated in cancer. Our workflow includes a pathologist review of the biopsy to ensure there is adequate amount of tumor for the assay followed by customized DNA extraction is performed on the specimen. Quality control of the specimen includes steps for quantity, quality and integrity and only after the extracted DNA passes these metrics an amplicon library is generated and sequenced. The resulting data is analyzed through an in-house bioinformatics pipeline and the variants are reviewed and interpreted for pathogenicity. Here we provide a snapshot of the utility of each panel using two clinical cases to provide insight into how a well-designed NGS workflow can contribute to optimizing clinical outcomes.

This manuscript describes clinical protocols for two next-generation sequencing panels. One panel interrogates hematologic malignancies while the other panel targets genes commonly mutated in solid tumors. Molecular classification of driver mutations in human malignancies offers valuable prognostic and predictive information.

新一代测序为可操作的突变在固体和液体瘤检测

As our understanding of the driver mutations necessary for initiation and progression of cancers improves, we gain critical information on how specific ...

科研

癌症研究

Wild-type Blocking PCR Combined with Direct Sequencing as a Highly Sensitive Method for Detection of Low-Frequency Somatic Mutations

Accurate detection and identification of low frequency mutations can be problematic when assessing residual disease after therapy, screening for emerging resistance mutations during therapy, or when patients have few circulating tumor cells. Wild-type blocking PCR followed by sequencing analysis offers high sensitivity, flexibility, and simplicity as a methodology for detecting these low frequency mutations. By adding a custom designed locked nucleic acid oligonucleotide to a new or previously established conventional PCR based sequencing assay, sensitivities of approximately 1 mutant allele in a background of 1,000 WT alleles can be achieved (1:1,000). Sequencing artifacts associated with deamination events commonly found in formalin fixed paraffin embedded tissues can be partially remedied by the use of uracil DNA glycosylase during extraction steps. The optimized protocol here is specific for detecting MYD88 mutation, but can serve as a template to design any WTB-PCR assay. Advantages of the WTB-PCR assay over other commonly utilized assays for the detection of low frequency mutations including allele specific PCR and real-time quantitative PCR include fewer occurrences of false positives, greater flexibility and ease of implementation, and the ability to detect both known and unknown mutations.

Wild-type blocking PCR followed by direct sequencing offers a highly sensitive method of detection for low frequency somatic mutations in a variety of sample types.

野生类型阻止PCR结合直接测序作为低频体细胞突变的检测的高灵敏度的方法

Accurate detection and identification of low frequency mutations can be problematic when assessing residual disease after therapy, screening for emerging ...

遗传学

A Novel Saturation Mutagenesis Approach: Single Step Characterization of Regulatory Protein Binding Sites in RNA Using Phosphorothioates

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to control gene expression. These regulatory proteins control gene expression either at the level of DNA (transcription) or at the level of RNA (pre-mRNA splicing, polyadenylation, mRNA transport, decay, and translation). Identification of regulatory sequences helps understand not only how a gene is switched on or off, but also which downstream genes are regulated by a particular regulatory protein. Here, we describe a one-step approach that allows saturation mutagenesis of a protein binding site in RNA. It involves doping DNA template with non-wild-type nucleotides within the binding site, synthesis of separate RNAs with each phosphorothioate nucleotide, and isolation of the bound fraction following incubation with protein. Interference from non-wild-type nucleotides results in their preferential exclusion from the protein-bound fraction. This is monitored by gel electrophoresis following selective chemical cleavage with iodine of phosphodiester bonds containing phosphorothioates (phosphorothioate mutagenesis or PTM). This single-step saturation mutagenesis approach is applicable to the characterization of any protein binding site in RNA.

Proteins that bind specific RNA sequences play critical roles in gene expression. Detailed characterization of these binding sites is crucial for our understanding of gene regulation. Here, a single-step approach for saturation mutagenesis of protein-binding sites in RNA is described. This approach is relevant for all protein-binding sites in RNA.

一种新的饱和诱变方法: 利用 Phosphorothioates 对 RNA 调控蛋白结合位点的单步表征

Gene regulation plays an important role in development. Numerous DNA- and RNA-binding proteins bind their target sequences with high specificity to ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Comparative Lesions Analysis Through a Targeted Sequencing Approach

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective anti-tumor strategies. Although concerns are frequently raised due to differences in sample processing and depth of coverage, next-generation sequencing of solid tumors have unraveled a highly variable degree of ITH across tumor types. Capturing the genetic relatedness between primary and metastatic lesions through the identification of clonal and subclonal populations is critical to the design of therapies for advance-stage diseases. Here, we report a method for comparative lesions analysis that allows for the identification of clonal and subclonal populations among different specimens from the same patient. The experimental approach described here integrates three well-established approaches: histological analysis, high-coverage multi-lesion sequencing, and immunophenotypic analyses. In order to minimize the effects on the detection of subclonal events by inappropriate sample processing, we subjected tissues to careful pathological examination and neoplastic cell enrichment. Quality controlled DNA from neoplastic lesions and normal tissues was then subjected to high coverage sequencing, targeting the coding regions of 409 relevant cancer genes. While only looking at a limited genomic space, our approach enables evaluating the extent of heterogeneity among somatic alterations (single-nucleotide mutations and copy-number variations) in distinct lesions from a given patient. Through comparative analysis of sequencing data, we were able to distinguish clonal vs. subclonal alterations. The majority of ITH is often ascribed to passenger mutations; therefore, we also used immunohistochemistry to predict functional consequences of mutations. While this protocol has been applied to a specific tumor type, we anticipate that the methodology described here is broadly applicable to other solid tumor types.

This article describes a method to identify clonal and subclonal alterations among different specimens from a given patient. Although the experiments described here focus on a specific tumor type, the approach is broadly applicable to other solid tumors.

通过有针对性的测序方法进行比较病变分析

Assessing intra-tumoral heterogeneity (ITH) is of paramount importance to anticipate failure of targeted therapies and design accordingly effective ...

行为学

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular signatures, or to evolve proteins in the laboratory. Here, we present a protocol that allows the generation of large (>1011) mutant libraries for a given target sequence. This method is based on replication of a ColE1 plasmid encoding the desired sequence by a low-fidelity variant of DNA polymerase I (LF-Pol I). The target plasmid is transformed into a mutator strain of E. coli and plated on solid media, yielding between 0.2 and 1 mutations/kb, depending on the location of the target gene. Higher mutation frequencies are achieved by iterating this process of mutagenesis. Compared to alternative methods of mutagenesis, our protocol stands out for its simplicity, as no cloning or PCR are involved. Thus, our method is ideal for mutational labeling of plasmids or other Pol I templates or to explore large sections of sequence space for the evolution of activities not present in the original target. The tight spatial control that PCR or randomized oligonucleotide-based methods offer can also be achieved through subsequent cloning of specific sections of the library. Here we provide protocols showing how to create a random mutant library and how to establish drug-based selections in E. coli to identify mutants exhibiting new biochemical activities.

Mutagenesis and Functional Selection Protocols for Directed Evolution of Proteins in E. coli

Here we demonstrate a simple protocol to create a random mutant library for a given target sequence. We show how this method, which is performed in vivo in Escherichia coli, can be coupled with functional selections to evolve new enzymatic activities.

导演蛋白质的演化突变和功能选择协议 E。大肠杆菌</em

The efficient generation of genetic diversity represents an invaluable molecular tool that can be used to label DNA synthesis, to create unique molecular ...

生物学

Competitive Genomic Screens of Barcoded Yeast Libraries

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily. The tempo of these advances is accelerating, promising greater depth and breadth. In light of these extraordinary advances, the need for fast, parallel methods to define gene function becomes ever more important. Collections of genome-wide deletion mutants in yeasts and E. coli have served as workhorses for functional characterization of gene function, but this approach is not scalable, current gene-deletion approaches require each of the thousands of genes that comprise a genome to be deleted and verified. Only after this work is complete can we pursue high-throughput phenotyping. Over the past decade, our laboratory has refined a portfolio of competitive, miniaturized, high-throughput genome-wide assays that can be performed in parallel. This parallelization is possible because of the inclusion of DNA 'tags', or 'barcodes,' into each mutant, with the barcode serving as a proxy for the mutation and one can measure the barcode abundance to assess mutant fitness. In this study, we seek to fill the gap between DNA sequence and barcoded mutant collections. To accomplish this we introduce a combined transposon disruption-barcoding approach that opens up parallel barcode assays to newly sequenced, but poorly characterized microbes. To illustrate this approach we present a new Candida albicans barcoded disruption collection and describe how both microarray-based and next generation sequencing-based platforms can be used to collect 10,000 - 1,000,000 gene-gene and drug-gene interactions in a single experiment.

We have developed comprehensive, unbiased genome-wide screens to understand gene-drug and gene-environment interactions. Methods for screening these mutant collections are presented.

条形码酵母图书馆竞争力的基因屏幕

By virtue of advances in next generation sequencing technologies, we have access to new genome sequences almost daily.  The tempo of these advances is ...

Detecting Somatic Genetic Alterations in Tumor Specimens by Exon Capture and Massively Parallel Sequencing

Efforts to detect and investigate key oncogenic mutations have proven valuable to facilitate the appropriate treatment for cancer patients. The establishment of high-throughput, massively parallel "next-generation" sequencing has aided the discovery of many such mutations. To enhance the clinical and translational utility of this technology, platforms must be high-throughput, cost-effective, and compatible with formalin-fixed paraffin embedded (FFPE) tissue samples that may yield small amounts of degraded or damaged DNA. Here, we describe the preparation of barcoded and multiplexed DNA libraries followed by hybridization-based capture of targeted exons for the detection of cancer-associated mutations in fresh frozen and FFPE tumors by massively parallel sequencing. This method enables the identification of sequence mutations, copy number alterations, and select structural rearrangements involving all targeted genes. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus conferring high sensitivity for detecting low frequency mutations.

We describe the preparation of barcoded DNA libraries and subsequent hybridization-based exon capture for detection of key cancer-associated mutations in clinical tumor specimens by massively parallel "next generation" sequencing. Targeted exon sequencing offers the benefits of high throughput, low cost, and deep sequence coverage, thus yielding high sensitivity for detecting low frequency mutations.

由外显子捕获和大规模平行测序检测体细胞遗传学改变在肿瘤标本

Efforts  to detect and investigate key oncogenic mutations have proven valuable to  facilitate the appropriate treatment for cancer patients. The ...

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an affinity purified yeast kinase fusion protein containing a V5-epitope tag for read-out. Purified kinase is obtained through culture of a yeast strain optimized for high copy protein production harboring a plasmid containing a Kinase-V5 fusion construct under a GAL inducible promoter. The yeast is grown in restrictive media with a neutral carbon source for 6 hr followed by induction with 2% galactose. Next, the culture is harvested and kinase is purified using standard affinity chromatographic techniques to obtain a highly purified protein kinase for use in the assay. The purified kinase is diluted with kinase buffer to an appropriate range for the assay and the protein microarrays are blocked prior to hybridization with the protein microarray. After the hybridization, the arrays are probed with monoclonal V5 antibody to identify proteins bound by the kinase-V5 protein. Finally, the arrays are scanned using a standard microarray scanner, and data is extracted for downstream informatics analysis1,2 to determine a high confidence set of protein interactions for downstream validation in vivo.

Using protein microarrays containing nearly the entire S. cerevisiae proteome is probed for rapid unbiased interrogation of thousands of protein-protein interactions in parallel. This method can be utilized for protein-small molecule, posttranslational modification, and other assays in high-throughput.

探讨高密度功能蛋白微阵列来检测蛋白质 - 蛋白质相互作用

High-density functional protein microarrays containing ~4,200 recombinant yeast proteins are examined for kinase protein-protein interactions using an ...

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on cancer, and especially cancer genetics. Current knowledge and future discoveries will make it necessary to study a huge number of genes that could be involved in a genetic predisposition to cancer. In this regard, we developed a Nextera design to study 11 complete genes involved in DNA damage repair. This protocol was developed to safely study 11 genes (ATM, BARD1, BRCA1, BRCA2, BRIP1, CHEK2, PALB2, RAD50, RAD51C, RAD80, and TP53) from promoter to 3&#39;-UTR in 24 patients simultaneously. This protocol, based on transposase technology and gDNA enrichment, gives a great advantage in terms of time for the genetic diagnosis thanks to sample multiplexing. This protocol can be safely used with blood gDNA.

gDNA Enrichment by a Transposase-based Technology for NGS Analysis of the Whole Sequence of BRCA1, BRCA2, and 9 Genes Involved in DNA Damage Repair

gDNA enrichment for NGS sequencing is an easy and powerful tool for the study of constitutional mutations. In this article, we present the procedure to analyse simply the complete sequence of 11 genes involved in DNA damage repair.

基因组DNA富集转座为基础的技术的整个序列的农工商分析<em&gt; BRCA1基因</em&gt;<em&gt; BRCA2</em&gt;和9个基因参与DNA损伤修复

The widespread use of Next Generation Sequencing has opened up new avenues for cancer research and diagnosis. NGS will bring huge amounts of new data on ...

经过一蛋白饱和诱变图书馆的功能评价规范利用高通量测序

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